A logic-gated DNA nanocascade for dual-RNA imaging in a triple-negative breast cancer cell model.

Triple-negative breast cancer (TNBC) is highly heterogeneous, and accurate molecular subtyping remains challenging because single biomarkers often lack sufficient specificity. Here, we developed an AND logic-gated DNA nanocascade for dual-RNA imaging of miR-21 and PD-L1 mRNA in TNBC cells. The system integrates a miR-21-activated DNAzyme, a PD-L1 mRNA-responsive entropy-driven strand displacement reaction (EDR), and hybridization chain reaction (HCR) amplification. The DNA circuit was encapsulated in Mn-Fe Prussian blue analogue (PBA) metal-organic frameworks and coated with homologous cancer cell membranes to form PBA@DNAzyme-EDR-HCR(DEH)@MDA-MB-231 cell membrane (MM) nanoprobes. The system exhibited detection limits of 0.7 pM for miR-21 and 0.5 pM for PD-L1 mRNA under excess complementary-input conditions and remained responsive when both targets were simultaneously present at 0.5 pM. In MDA-MB-231 cells, simultaneous recognition of both RNA inputs produced strong fluorescence, whereas inhibition of either target markedly reduced the signal. Additional experiments in HCC1806 and AU565 cells further supported differential fluorescence responses across the tested breast cancer cell models. These results demonstrate an AND logic-gated strategy that operates without exogenous protein enzymes and combines dual-RNA recognition with cascade amplification for molecular subtype discrimination.